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Title: A modified reverse one-hybrid screen identifies transcriptional activation in Phyochrome-Interacting Factor 3

Author
item DALTON, J.C. - University Of California
item BATZ, U - University Of California
item LIU, J - University Of California
item CURIE, G.L. - University Of California
item QUAIL, P.H. - University Of California

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/2/2014
Publication Date: 10/21/2014
Citation: Dalton, J., Batz, U., Liu, J., Curie, G., Quail, P. 2014. A modified reverse one-hybrid screen identifies transcriptional activation in Phyochrome-Interacting Factor 3. Frontiers in Plant Science. 7:881.

Interpretive Summary: This paper reports the development of a new method for identifying mutations in transcription factors that cause loss of the capacity to induce gene expression. We used of this method to identify transcriptional activation domains in a quartet of bHLH-class transcription factors called PIF1, PIF3, PIF4 and PIF5 (for Phytochrome Interacting Factors) and found two such domains within these proteins. In addition to providing insight into the mechanism by which these factors may activate target gene expression, this study will provide a valuable tool for researchers interested in screening for PIF-interacting proteins.

Technical Abstract: Transcriptional activation domains (TAD) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5' -phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negative selection that can be used for both reverse-1- and 2-hybrid screens. With this method we were able to identify two distinct regions in PIF3 with transcriptional activation activity, both of which are functionally conserved in PIF1, PIF4, and PIF5. Both are collectively necessary for full PIF3 transcriptional activity, but neither is sufficient to induce transcription autonomously. We also found that the TAD appear to overlap physically with other PIF3 functions, such as phyB binding activity and consequent phosphorylation. Our protocol should provide a valuable tool for identifying, analyzing and characterizing novel TAD in eukaryotic transcription factors, and thus potentially contribute to the unraveling of the mechanism underlying transcriptional activation.